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RESEARCH Open Access Endothelial-like cells in chronic thromboembolic pulmonary hypertension: crosstalk with myofibroblast-like cells Seiichiro Sakao 1* , Hiroyuki Hao 2 , Nobuhiro Tanabe 1 , Yasunori Kasahara 1 , Katsushi Kurosu 1 and Koichiro Tatsumi 1 Abstract Background: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by intravascular thrombus formation in the pulmonary arteries. Recently, it has been shown that a myofibroblast cell phenotype was predominant within endarterectomized tissues from CTEPH patients. Indeed, our recent study demonstrated the existence of not only myofibroblast-like cells (MFLCs), but also endothelial-like cells (ELCs). Under in vitro conditions, a few transitional cells (co-expressing both endothelial- and SM-cell markers) were observed in the ELC population. We hypothesized that MFLCs in the microenvironment created by the unresolved clot may promote the endothelial-mesenchymal transition and/or induce endothelial cell (EC) dysfunction. Methods: We isolated cells from these tissues and identified them as MFLCs and ELCs. In order to test whether the MFLCs provide the microenvironment which causes EC alterations, ECs were incubated in serum-free medium conditioned by MFLCs, or were grown in co-culture with the MFLCs. Results: Our experiments demonstrated that MFLCs promoted the commercially available ECs to transit to other mesenchymal phenotypes and/or induced EC dysfunction through inactivation of autophagy, disruption of the mitochondrial reticulum, alteration of the SOD-2 localization, and decreased ROS production. Indeed, ELCs included a few transitional cells, lost the ability to form autophagosomes, and had defective mitochondrial structure/ function. Moreover, rapamycin reversed the phenotypic alterations and the gene expression changes in ECs co- cultured with MFLCs, thus suggesting that this agent had beneficial therapeutic effects on ECs in CTEPH tissues. Conclusions: It is possible that the microenvironment created by the stabilized clot stimulates MFLCs to induce EC alterations. Keywords: neointima, myofibroblast, endothelial cells, CTEPH. Background It is generally known that chronic thromboem bolic pul- monary hypertension (CTEPH) is one of the leading causes of severe pulmonary hypertension. CTEPH is characterized by intravascular thrombus formation and fibrous stenosis or complete obliteration of the pulmon- ary arteries [1]. The consequence is increased pulmon- ary vascular resistance, resulting in pulmonary hypertension and progressive right heart failure. Pulmonary endarterectomy (PEA) is the current main- stream of therapy for CTEPH [2]. Moreover, recent stu- dies have provided evidence suggesting that, although CTEPH is believed to result from acute pulmonary embolism [3,4], small-vessel disease appears a nd wor- sens later in the course of disease [5]. Histopathologic studies of microvascular changes in CTEPH have shown indistinguishable vascular lesions from those seen in idiopathic pulmonary arterial hypertension (IPAH ) and Eisenmenger’s syndrome [6-8]. Especially in vitro and ex vivo experiments, pulmonary artery endothelial cell (EC) in the group of pulmonary hypertensive diseases are suggested to exhibit an unusual hyperproliferative * Correspondence: sakaos@faculty.chiba-u.jp 1 Department of Respirology (B2), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan Full list of author information is available at the end of the article Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 © 2011 Sakao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), w hich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. potential with decreased susceptibility to apoptosis [9,10], indicating that dysfunctional EC may contribute to the progression of the diseases. Recently, Firth et al showed that multipotent mesenchymal progenitor cells are present in endarterec- tomized tissues from patients with CTEPH, and that a myofibroblast cell phenotype was predominant within these tissues, contributing extensively to the vascular lesion/clot [ 11]. Indeed, we have a lso demonstrated the existence of not only myofibroblast-like cells (MFLCs), but also endothelial-like cells (ELCs) in these tissues [12]. Under in vitro conditions, morphological altera- tions were more easily detected in the ELCs. Smooth muscle (SM)-like cel ls (defined by their expression of a- SM-actin (SMA)) and a few transitional cells (co-expres- sing b oth endothelial- (von Willebrand factor) and SM- (a-SMA) cell markers) were consistently observed by immunohistochemical staining (preliminary data). In vitro experiments conducted to ass ess the contribu- tion of ECs to the development of pulmonary arterial hypertension (PAH) have demonstrated that the shift to a transdifferentiated phenotype could be attributed to selection of distinct cell subpopulations (i.e., stem-like cells). These findings also suggest that the endothelial- mesenchymal transition (EnMT) might be an important contributor to pathophysiological vascular remodeling in the complex vascular l esions of PAH [13], because, although bone marrow-derived cells could participate in arterial neointimal formation after mechanical injury, they did not contribute substantially to pulmonary arter- ial remodeling in an experimental PAH model [14]. Autophagy is a catabolic process involving the degra- dation of intracellular material that is evolutionarily con- served between all eukaryotes. During autophagy, cytoplasmic components are engulfed by double-mem- brane-bound structures (autophagosome s) and delivered to lysosomes/vac uoles for degradation [15]. Recent stu- dies indicate that autophagy plays an important role in many different pathological conditions. Indeed, both activation and inactivation of autophagy may impact cancer cell growth. If autophagy cannot be activated, protein synthesis predominates over protein degrada- tion, and tumor growth is stimulated. In contrast, autop- hagy may be activated in more advanced stages of cancer to guarantee the survival of cells in minimally- vascularized tumors [16]. The interactions between ECs and smooth muscle cells (SMCs), which exist in close contact via a func- tional syncytium, are involved in the process of new ves- sel formation that occurs during development, as part of wound repair, and during the reproductive cycle [17-19]. We hypothesized that MFLCs stimulated by the microenvironment created by the unresolved clot may promote ECs to transit to other mesenchymal phenotypes and/or induce EC dysfunction, contributing to the vascular lesion, i.e., not only proximal vasculature, but also microvascular. In the experiments considered here, we isolated cells from endarterectomized tissue from patients with CTEPH and id entified them as MFLCs and ELCs. In order to show the hypothesis, human pulmonary microvascular ECs were incubated in a serum-free medium conditioned by MFLCs, or ECs were co-cultured with M FLCs. The aim of this study was to examine whether MFLCs in the microenviron- ment created by the unresolved clot can, in principle, affect EC disorder through the EnMT and autophagy. Methods Cell lines and reagents The PEA tissues of patients with CTEPH were obtained following PEA performed by Dr. Masahisa Masuda at the Chiba Medical Center, Japan. Control pulmonary arteries were obtained following lung resection for per- ipheral cancer by Dr. Ichiro Yoshino at the Chiba Uni- versity Hospital, Japan. Written informed co nsent was acquired before surgery from all patients from whom tissue samples were obtained. The study was app roved by the Research Ethics Committee of Chiba University School of Medicin e, and all subjects gave their informed consent in writing. Although not clinica lly accurate, the PEA tissues were defined as mentioned below. PEA samples obtained from the region directly surrounding the fibrotic clot are referred to as “proximal” vascular tissue and those obtained from areas after the fibrotic clot region are referred to as the “distal” vascular tissue [11]. The tissues were cultured and various explant out- growth cells were dissociated as described previously [12]. Myofibroblast-like cells (MFLCs) and endothelial- like cells (ELCs) were isolated and identified from endarterectomized tissue from patients with CTEPH and pulmonary arterial fibroblast-like cells from control pulmonary arteries. PEA samples obtained from a total of six patients undergoing PEA were examined in this study. Human pulmonary micr ovascular ECs w ere obtained from Lonza Inc (Allendale, NJ, USA). T he following ant ibod ies were used during our present studies: mouse anti-a-SMA (1:1000, Sigma, St. Louis, MO, USA), mouse anti-vimentin (1:200, DAKO, Carpinteria, CA, USA), mouse anti-human desmin (1:100, DAKO, Car- pinteria, CA, USA), anti-mouse IgG Ab conjugated with Rhodamine dye (1:500, Molecular Probes, Eugene, OR, USA), rabbit anti-von Willebrand factor (Factor VIII) (1:1000, DAKO, Carpinteria, CA, USA), anti-rabbit IgG conjugated with Alexa-488 fluor escent dye (1:500, Mole- cularProbes,Eugene,OR,USA),andrabbitanti-CD31 (1:1000, DAKO, Carpinteria, CA, USA). Rapamycin was purchased from Merck (Frankfurter, Germany). Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 2 of 16 Immunofluorescence staining The cells were fixed in a 1:1 mixture of methanol and acetone for 2 minutes followed by blocking with normal goat serum for 30 minutes as described previously [13]. The cells were incubated with primary antibodies (anti- a-smooth muscle actin (SMA), anti-von Willebrand fac- tor, anti-vimentin and anti-desmin) for 1 hour at room temperature, and then with secondary antibodies (anti- mouse IgG conjugated with Alexa-594 fluorescent dye and anti-rabbit I gG conjugated with Alexa-488 fluores- cent dye) for 1 hour at room temperature. Stained cells were embedded in VectaShield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) and were examined with a NIKON Eclipse 80 i microscope (Nikon, Tokyo, Japan) using the VB-7210 imaging sys- tem (Keyence, Tokyo, Japan). Positive cells were counted in 3 different fields at a magnification of × 200 using a fluorescence microscope. Double immunohistochemical staining Endarterectomized samples were embedded in optimal cutting temperature (OCT) compound (Sakura Tissue Tek), frozen, and cut into 10- μm sections with a cryo- stat. For basic characterization, standard hematoxylin and eosin ( H & E) staining was per formed. The CD31 antibody was used to stain endarterectomized tissue, together with aSMA to stain transitional cells. aSMA staining (blue) was developed with alkaline phosphatase- conjugated secondary antibody, and then CD31 staini ng (brown) was developed with peroxidase-conjugated sec- ondary antibody. Transitional cells were confirmed by aSMA posit ively stained cytosol that also had concomi- tant positive cytoplasmic staining in CD31 positive cells. ELISA (Enzyme-Linked ImmunoSorbent Assay) TGF-b 1 were measured by sandwich ELISA techniques by ELISA Tech (Aurora, CO, USA) utilizing reagents from R&D systems (Minneapolis, MN, USA). The sam- ples were read in a spectrophotometer at 405 nm. Anti- bodies and tracer were bought from C ayman Chemicals (Ann Arbor, Mi, USA). Human pulmonary microvascular ECs in the conditioned medium At passage 2 MFLCs or pulmonary arterial fibroblast- like cells were seeded at a density of 1.5 × 10 4 cells/ cm 2 and were subcultured when they were to 90% con- fluences (4-8 days). They were washed 3 times using phosphate-buffered saline (PBS) and were incubated with serum-free medium for 48 hours. HPMVEC were seeded in 6 cm dishes at 1 × 10 5 density and cultured in EGM supplemented with 5% fetal bovine serum. At 70 to 80% confluence they were washed 3 times with PBS, incubated in the conditionedmediumfor48hoursand incubated in EGM again for 48 hours. After the incuba- tion periods, they were assessed microscopically, further characterized by immunohistochemical staining and har- vested to extract RNA for quantitative RT-PCR and to extract protein for ELISA. Co-culture of human pulmonary microvascular ECs and MFLCs Co-culture of human pulmon ary microvascular ECs and MFLCs was done on a 6-well plate (BD Falcon) with Cell Culture Inserts (Falcon, 353102, 1.0 microns pore size). Human pulmonary microvascular ECs or pulmon- ary arterial fibroblast- like cells (at 5 × 10 4 density) and MFLCs (at 5 × 10 4 density) were added into the lower or upper chamber with or without rapamycin (10 nM). After tw o weeks incubation periods, they were assessed microscopically and further characterized by immuno- histochemical staining, harvested to extract RNA for PCR array, and other assays. Magnetic cell sorting (MACS) After trypsinization of ECLCs at passage 2, CD31 posi- tive cells w ere isol ated by using CD31 MicroBeads (Direct CD31 progenitor cell isolation kit, Miltenyi Bio- tec Inc, A uburn, CA, USA) as described previously [13]. After trypsinization of ECLCs at passage 2, 100 μlof FcR Blocking Reagent (Direct CD31 progen itor cell iso- lation kit, Milte nyi Biotec Inc, Auburn, CA, USA) per 10 8 total cells was added to the cell suspension to inhi- bit nonspecific or Fc-receptor mediated binding of CD31 MicroBeads (Direct CD31 progenitor cell isolation kit, Miltenyi Biotec) to non-target cells. Cells were labeled by adding 100 μl CD31 MicroBeads per 10 8 total cells, and incubated for 30 min at 6-12°C. After washing, cells were resuspended in 500 μl buffer and applied to the MS+/RS+ column with the column adapter in the magnetic field of the MACS separator. The co lumn was washed 3× with 500 μl buffer. The column was removed from the separat or and t he retained cells were flushed out with 1 ml buffer under pressure using the plunger supplied with the column. The cells were incubated in EGM and cultured until passage 5. Total RNA isolation and Quantitative measurement Total RNA was extracted from human pulmonary microvascular ECs with an RNeasy Mini Kit (Qi agen, CA, USA). RNA and cRNA yields were quantitated on a Nano-Drop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) as described previously [13]. PCR array analysis RT 2 Profiler™ PCR Arrays ( SABiosciences, Frederick, USA) are the reliable and sensitive tools for analyzing Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 3 of 16 the expression of a focused panel of genes in signal transduction pathways, biological process or disease related gene networks. The 96-well plate Human Autop- hagy PCR-array (PAHS-084) which profiles the expres- sion of 84 key genes involved in autophagy and Human Endothelial Cell Biology PCR-array (PAHS-015) which profiles the expression of 84 genes related to endothelial cell biology were selected as the hypothesis. There is a better sensitivity of quantitative PCR in comparison to microarray [20,21]. The PCR Arrays can be used for research on various disease including cancer, immunology, and phenotypic analysis of cells. The mRNA of each co-cultured EC was converted into cDNA using the RT 2 First Strand Kit (SABios- ciences, Frederick, USA). This cDNA was then added to the RT 2 SYBR Green qPCR Master Mix (SABiosciences, Frederick, USA). Next, each sample was aliquotted on PCR-arrays. All steps were done according to the man u- facturer’ s protocol for the ABI Prism 7000 Sequence Detection System. To analyze the PCR-array data, an MS-Excel sheet with macros was downloaded fro m the manufacturer’s website http://www.sabiosciences.com/ pcrarraydataanalysis.php. The website also allowed online analysis. For each PCR reaction, the excel sheet calculated two normalized average C t values, a paired t test P value and a fold change. Data normalization was based on correcting all C t values for the average C t values of several constantly expressed housekeeping genes (HKGs) present on the array. PCR-array analysis results were evaluated. SMAD reporter assay TheSMADreporterassaydetectstheactivityofTGFb signaling pathway through monitoring the SMAD tran- scriptional response in cultured cells. Cignal SMAD Reporter (GFP) Kit (SABiosciences, Frederick, USA) was adapted to assess the activity of this signaling pathway. Co-cultured human pulmonary microvascular ECs with pulmonary arterial fibroblast-like cells or MFLCs were trypsinized, suspended at 1 × 10 4 /well at density, and seeded into 96-well cell culture plates. Transfection complexes including the signal reporters were aliquoted into wells containing overnight cell cultures. After 40 hours of transfection, expression of the GFP reporter was monitored via the fluorometry (Infinite 200 PRO, Tecan Group Ltd., Männedorf, Switzerland). All steps were done according to the manufacturer’s protocol. Reactive oxygen species (ROS) assay Mea suring ROS activ ity intracellularly, we adapted Oxi- Select ROS assay kit (Cel l Biolabs, Inc., San Diego, USA). Co-cultured human pulmonary microvascular ECs with pulmonary arterial fibroblast-like cells or MFLCs were trypsinized, suspended at 1 × 10 4 /well at density, and seeded into 96-well cell culture plates. Media was removedfromallwellsandcellswerewashedwith DPBS 3 times. 100 μL of 1 × 2,7-dichlorofluorescein dia- cetate (DCFH)-DA/media solution added to cells and they were incubated at 37 ° for 60 minutes. Solution was removed and cells were washed with DPBS 3 times. DCFH-DA loaded cells were t reated with hydrogen per- oxide (100 μM) in 100 μL medium. After 1 hour, the fluorescence was read via the fluorometry (Infinite 200 PRO, Tecan Group Ltd., Männedorf, Switzerland). All steps were done according to the manufacturer’ s protocol. Statistical analysis Three independent experiments were performed and subjected to statistical analysis. T he results were expressed as the means ± SEM. PCR array data were analyzed using a paired t test according to the manufac- turer’s protocol and othe r data were the Mann-Whitney U test. A p < 0.05 was considered to be significant for all comparisons. Results The cellular composition of endarterectomized tissue from CTEPH patients Two different cell types were isolated from the “distal” vascular tissue in the patients with CTEPH. The cell typesweredeterminedbymorphologytobeELCs (rounded appearance and cell-cell contact in the mono- layer) and MFLCs (spindle-shaped with cytoplasmic extensions) (Figure 1). They were dissociated and pas- saged free from surrounding cells using cloning cylin- ders. MFLCs were prepared from each of the six patients and ELCs could be isolated from 4 of the six patients. Figure 1 Cells from endarterectomized tissue. The MFCsL and ELCs from endarterectomized tissue were microscopically assessed. The magnification was 100×. Scale bar = 100 μm; MFLCs = myofibroblast-like cells; ELCs = endothelial-like cells. Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 4 of 16 Moreover, another cell type was isolated from the ves- sel wall tissues of control pulmonary arteries, defined morphologically as fibroblast-like cells (pulmonary arter- ial fibroblast-like cells) (data not shown). These cells were used as control cells, and were prepared in the same way as the CTEPH specimens. The cells outgrown from the organized thrombotic tis- sue and control pulmonary arteries were further charac- terized by immunohistochemical staining for desmin, vimentin, von Willebrand factor (Factor VIII) anda- SMA. ELCs were positively stained for the endothelial cell (EC)-specific marker (Factor VIII) and the mesenchy- mal-specific marker (vimentin) and negative for the 2 smooth muscle cell (SMC)-specific markers (desmin and a-SMA) [12]. MFLCs were Factor VIII and desmin nega- tive and vimentin and a-SMA positive [12]. Pulmonary arterial fibroblast-like cells were Factor VIII, desmin and a-SMA negative, and vimentin positive (data not shown). Phenotypic alteration of ELCs After a few passages, morphological alterations were detected in the ELCs. The cell-cell contact of the endothelial monolayers became disrupted, and some ELCs had lost their rounded appearance and acquired an elongated, mesenchymal-like morphology. At the 2nd passages, the morphological alterations could not to be detected micr oscopically (Figure 2A), but some SM-like cells (as defined by expression of a-SMA) (Figure 2B) and a few transitional cells (co-expressing both endothe- lial- and SM-cell markers) were consistently observed (Figure 2C) by immunohistochemical staining. These transitional cells could be observed in ELCs prepared from 4 of the six samples. Since this result suggested that ELCs were contami- nated with SMCs, at the 3rd passage, they were sorted for the EC marker CD31 in order to establish that the ELCs were free of contamination with SMCs. After magnetic cell sorting for the EC marker CD31, ELCs were examined microscopically, and unusual “ pile” growth and disrupted formation of the endothelial monolayer were detected (Figure 2D). Moreover, SM- like cells (Figure 2E) and transitional cells were consis- tently observed (Figure 2F). Transitional cells in endarterectomized CTEPH tissue To detect transitional cells which co-express both endothelial (CD31) and SM (a-SMA) markers in the PEA tissues of patients with CTEPH, a double immu- nostaining method for CD31/a-SMA was performed. The HE staining of the neointimal layers of both the “proximal” and the “distal” vascular tissues indicated the presence of a fibrin network, and nuclei are seen within this region (Figure 2G). These neointimal layers are composed of some a-SMA positive cells (Figure 2H). Although the neointimal layers of both the “ proximal” and the “distal” vascular tissues were composed of a- SMA positive cells, CD31 positive cells were found in the “distal” vascular wall tissue but not in the “proximal” vascular tissue (Figure 2I). As shown in Figure 2J, a few CD31 and a-SMA double-positive cells were identified in the “distal” vascular tissues, thus indicating the pre- sence of “ intermediate” cells, which were intermediate between ECs and muscle cells in structure, in the neoin- timal lesions of CTEPH patients. Decreased expression of Autophagic marker LC3 (microtubule-associated protein1 light chain 3; MAP1LC3), abnormal mitochondria, and decreased expression of superoxide dismutase (SOD)-2 in ELCs To assess ELC alterations, an immunofluorescence staining method for LC3, mitochondrial mark er mito- tracker red, and SOD-2 was performed. LC3 is a major constituent of the autophagosome, a double-membrane structure that sequesters the target organelle/protein and then fuses with endo/lysosomes where the contents and LC3 are degraded. Confocal microscopy showe d that th e ELCs did not express LC3. The formation of autophagosomes (green punctate structures) was not detected in these cells (Figure 2K). SOD-2 is an enzyme that catalyzes the dissociation of superoxide into oxygen and hydrogen peroxide. As such, this is an important antioxidant defense in nearly all cell s exposed to oxygen and i s located in the mitochon- dria. Immunofluorescence staining for mitochondrial marker mitotracker red revealed that the normal fila- mentous mitochondrial reticulum was disrupted and rarefied in ELCs (Figure 2L). Moreover, SOD-2 was decreased in ELCs (Figure 2M). Phenotypic alteration of human pulmonary microvascular ECs is induced by MFLCs-conditioned medium As mentioned above, ELCs isolated from the PEA tis- sues could easily change their phenotype during passa- ging. We postulated that the interactions of ELCs and MFLCs, which exist in close contact in the PEA tissues, are involved in a process of o rganized thrombus forma- tion that occurs during the development of CTEPH. One basic component of this interaction may be the MFLC-induced transition of ELCs. To test this hypoth- esis, the commercially available human pulmonary microvascular ECs were incubated in serum-free med- ium conditioned by MFLCs to determine whether MFLCs release m ediators which cause phenotypic alteration of human pulmonary microvascular ECs. We first established that the human pulmonary micro- vascular ECs were free of contamination with vascular smooth muscle cells (VSMCs) by morphology (rounded appearance and cell-cell contact of the monolayer) Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 5 of 16 (Figure3A)andbyimmunofluorescencestainingusing anti-von Willebrand factor (Figure 3D), anti-a-SMA (Figure 3D), anti- vimentin (data not shown), and anti- human desmin (data not shown) antibodies. The endothelial cell-specific marker and the mesenchymal- specific marker were positive, and the 2 smooth muscle- specific markers were negative, providing evidence that the human pulm onary microvascular ECs were not con- taminated with VSMCs. At the 2nd passage after incubation in serum-free medium conditioned by pulmonary arterial fibroblast- like cells and MFLCs, the phenotypic alteration of human pulmonary microvascular ECs was assessed microscopically and by immunofluorescence staining. The cell-ce ll contact of the endothelial mo nolayers became disrupted, and many ECs had lost their rounded appearance and acquired an elon gated, mesenchymal- like morphology in the medium conditioned by MFLCs (Figure 3C) in comparison to the medium conditioned by pulmonary a rterial fibroblast-like cells (Figure 3B). The number of ECs (as defined by ex pression of von Willebrand factor) decreased, and SM-like cells (as defined by expression of a-SMA) were consistently obs erved in the medium conditioned by MFLCs (Figure 3F, G), but not in the medium conditioned by pulmon- ary arterial fibroblast-like cells (Figure 3E, G). Expression of TGF-b1 protein in the conditioned medium Because TGF-b1 is known to be involved in inducing the endothelial-mesenchymal transition [22] and is known to promote a-SMA expression in non-muscle cells (ECs and fibroblasts derived from various t issues) [23,24], the protein levels in the conditioned medium were measured by ELISA. Serum-free medium conditioned by MFLCs contained higher TGF-b1 levels than medium condi- tioned by pulmonary arterial fibroblast- like cells, but the difference was not statistically significant (Figure 3H). Phenotypic alteration of human pulmonary microvascular ECs co-cultured with MFLCs After a 14 day incubation period, morphological altera- tions were detected in human pulmonary microvascular Figure 2 ELCs from endarterectomized tissue. A-F), ELCs were assessed by immunofluorescence staining for anti-Factor VIII (green) and anti- a-SMA (red) to confirm the phenotypes of the cells. A), B) and C), ELCs before sorting; D), E) and F), ELCs after sorting; A) and D), the magnification was 100×. Scale bar = 100 μm; B) and E), the magnification was 200×. Scale bar = 50 μm; C) and F), the magnification was 400×. Scale bar = 25 μm. The blue staining was DAPI. G-J), Immunohistochemical staining of endarterectomized tissue. The neointimal layer of distal vascular wall tissues was assessed by immunohistochemical staining. G), Hematoxylin and Eosin (HE) staining; H), Single staining for a-SMA; I), Single staining for CD31; J), Double staining for CD31 and a-SMA; the magnification was 200×. Scale bar = 50 μm. K, L, M), Immunofluorescence staining of ELCs for the autophagic marker, LC3 (K), mitochondrial marker mitotracker red (L), and SOD-2 (M). K), The formation of autophagosomes (green punctate structures) was not detected. L), The normal filamentous mitochondrial reticulum (red punctate structures) was not detected. M), SOD-2 expression (green punctate structures) was not detected. The blue staining was DAPI. The magnification was 400×. Scale bar = 25 μm. ELCs = endothelial-like cells. Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 6 of 16 Figure 3 Human pulmonary microvascular ECs (HPMVECs) in serum-free medium conditioned by pulmonary arterial fibroblast-like cells (PAFLCs) or myofibroblast-like cells (MFLCs). The phenotypic alteration of HPMVECs was assessed microscopically and by immunofluorescence staining. A) and D), Before incubation in serum-free medium conditioned by PAFLCs and MFLCs; B) and E), At the 2nd passage after incubation in serum-free medium conditioned by PAFLCs; C) and F), At the 2nd passage after incubation in serum-free medium conditioned by MFLCs; A), B) and C), microscopic findings; the magnification was 100×. Scale bar = 100 μm; D), E) and F), Immunofluorescence staining for anti-Factor VIII (green) and anti-a-SMA (red). The blue staining was DAPI. The magnification was 200×. Scale bar = 50 μm. F), Some cells were positive for smooth muscle actin fibers (see inset); HPMVECs = human pulmonary microvascular endothelial cells; MFLCs = myofibroblast like cells; PAFLCs = fibroblast-like cells from control pulmonary arteries. G) Positive cells for anti-von Willebrand factor and anti-a- SM-actin were counted in 3 different fields at a magnification of × 200 in a fluorescence microscope. *P < 0.05 VS. PAFLCs, n ≥ 3. H) The TGF-b1 protein levels in the conditioned medium were measured by ELISA. There were no significant differences between the serum-free medium conditioned by PAFLCs and MFLCs. Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 7 of 16 ECs co-cultured with MFLCs (Figure 4B, D), but not those cultured with pulmonary arterial fibroblast-like cells (Figure 4A, C). The cell-cell contact of the endothelial monolayers (Figure 4A) became disrupted, and hill and valley formation appeared. Moreover, some ECs had lost their rounded appearance and acquired an elongated, mesenchymal-like morphology (Figure 4B). Some SM-like cells (as defined by their expression of a- SMA) and a few transitional cells (co-expressing both endothelial- and SM- cell markers) were consistently observed (Figure 4D, E) by immunohistochemical staining. Autophagy PCR array analysis of human pulmonary microvascular ECs co-cultured with MFLCs There were decreases in the expression of 17 autop- hagy-related genes in ECs co-cultured with MFLCs in comparison to the expression in ECs co-cultured with pulmonary arterial fibroblast-like cells (Figure 5A) (Table 1). Four of these genes; AMBRA1, ATG4D, MAP1LC3B, and RGS19, are involved in autophagic vacuole formation. In particular, ATG4D is responsi- ble for protein targeting to the membrane/vacuole, and is responsible for protein transport and protease activity. Ten of the 17 genes; BCL2, BID, CDKN2A, CTSB, HSP90AA1, HTT, IFNG, IGF1, INS, and PRKAA1 are co-regulators of autophagy and apopto- sis. Three genes; RPS6KB1, TMEM77, and UVRAG are related to autophagy in response to other intracel- lular signals. Autophagic marker LC3 expression in human pulmonary microvascular ECs co-cultured with MFLCs Confocal microscopy showed that the ECs co-cultured with pulmonary a rterial fibroblast-like cells expressed LC3. The formation of aut ophagosomes (green punctate structures) was detected in these cells (Figure 6A), but not in E Cs co-cultured with MFLCs (Figure 6B) no r in ELCs (Figure 2K). Abnormal mitochondria and decreased expression of superoxide dismutase (SOD)-2 in human pulmonary microvascular ECs co-cultured with MFLCs Immunofluorescence staining for mitochondrial marker mitotracker red revealed that the normal filamentous mitochondrial reticulum observed in ECs co-cultured with pulmonary arterial fibroblast-like cells (Figure 6D) was disrupted and rarefied in both ECs co-cultured with MFLCs (Figure 6E) and ELCs (Figure 2L). Moreover, SOD-2 was decreased in ECs co-cultured with MFLCs (Figure 6H) and ELCs (Figure 2M) compared to those co-cultured with pulmonary arterial fibroblast-like cells (Figure 6G). T he decrease in SOD-2 expression in ECs co-cultured with MFLCs and ELCs might be associated with a reduction in SOD-2 activity. Endothelial cell biology PCR array of human pulmonary microvascular ECs co-cultured with MFLCs These results, including the phenotypic alterations, inac- tivation of autophagy, and mitochondrial dysfunction, suggested that the endothelialcellbiologyisalteredin patients with CTEPH. Therefore, an endothelial cell biology PCR array was done to fur ther explore the effects of MFLCs on endothelial cell biology. Figure 4 Human pulmonary microvascular ECs (HPMVECs) co- cultured with pulmonary arterial fibroblast-like cells (PAFLCs) or myofibroblast-like cells (MFLCs). The phenotypical alteration of HPMVECs was assessed microscopically and by immunofluorescence staining after a 14 day incubation period. A) and C), HPMVECs co- cultured with PAFLCs; B) and D), HPMVECs co-cultured with MFLCs; A) and B), Microscopic findings; the magnification was 100×. Scale bar = 100 μm; C) and D), Immunofluorescence staining for anti- Factor VIII (green) and anti-a-SMA (red). The blue staining was DAPI. The magnification was 400×. Scale bar = 25 μm. D), Some cells coexpressed both anti-Factor VIII and anti-a-SMA (see inset); HPMVECs = human pulmonary microvascular endothelial cells; MFLCs = myofibroblast-like cells; PAFLCs = fibroblast-like cells from control pulmonary arteries. E) Positive cells for anti-von Willebrand factor and anti-a-SM-actin were counted in 3 different fields at a magnification of × 200 in a fluorescence microscope. *P < 0.05 VS. PAFLCs, n ≥ 3. Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 8 of 16 There were decreases in the expression of 15 and increases in the expression of 3 genes in ECs co-cul- tured with MFLCs in comparison to the expression in those co-cultured with pulmonary arterial fibroblast-like cell s (Figure 5B). The 15 decreased genes were ANXA5, BCL2, CDH5, COL18A1, CX3CL1, ITGA5, ITGAV, ITGB1, MMP1, NPPB, PGF, PLA2G4C, PLAU, RHOB, and SOD1 (Table 2). C DH5, COL18A1, CX3CL1, ITGA5, ITGAV, ITGB1 and RHOB are related to endothelial cell activation as adhesion molecules. MMP1, NPPB, PLAU and RHOB are related to endothe- lial cell activation, and are part of the extracellular matrix (ECM) molecules. ANXA5 and PLAU are related to endothelial cell activation with regard to thrombin activity. PGF is related to angiogenesis. PLA2G4C and SOD-1 are both related to the endothelial cell response to stress. The 3 genes with increased expression were AGTR1, CASP1, and TIMP1 (Table 3). AGTR1 is related to the permissibility and vessel tone of the angiotensin system. CASP1 is related to endothelial cell injury and resulting apoptosis. TIMP1 is related to endothelial cell activation and cell growth. SMAD reporter signal in human pulmonary microvascular ECs co-cultured with MFLCs The SMAD2 and SMAD3 proteins are phosphorylated and activated by TGF-b signaling. These activ ated SMAD 2 a nd SMAD 3 t hen form complexes with the SMAD4. These SMAD complexes then migrate to the nucleus, where they activate the expression of TGF-b- responsive genes. Besides simple concentration measurements of TGF- b1 in the conditioned medium (Figure 3H), the activa- tion of the TGF-b signaling in human pulmonary micro- vascular ECs co-cultured with MFLCs were measured by the SMAD reporter assay. There was no statistical dif- ference in the expression of SMAD reporter signal in ECs co-cultured with MFLCs in comparison to the expression in those co-cultured with pulmonary arterial fibroblast-like cells (Figure 5C). Accumulation of ROS in human pulmonary microvascular ECs co-cultured with MFLCs Accumulation of ROS coupled with an increase in oxi- dative stress has been implicated in the pathogenesis of numerous disease states. As SOD1 and SOD2 downre- gulation have been shown by the PCR-Arrays (Figure 5B) and immunofluorescence (Figure 6H), the missing production of ROS might be involved in ECs co-cul- turedwithMFLCs[25].Thedecreasedproductionof ROS has been detected in ECs co-cultured with MFLCs in comparison to the expression in those co-cultured with pulmonary arterial fibroblast-like cells (Figure 5D). Rapamycin treatment Prolonged rapamycin treatme nt of ECs co-cultu red with MFLCs reversed the decrease in the 17 autophagy- Figure 5 Human pulmonary microvascular ECs (HPMVECs) co- cultured with pulmonary arterial fibroblast-like cells (PAFLCs) or myofibroblast-like cells (MFLCs). Autophagy and Endothelial cell biology. A) Autophagy PCR array analysis of HPMVECs co- cultured with PAFLCs, MFLCs or MFLCs+Rapamycin. There were decreases in the expression of 17 autophagy-related genes in the ECs co-cultured with MFLCs in comparison those co-cultured with PAFLCs (P < 0.05; n = 3). This result is related to 3 different patients out of six of co-culture or conditioned medium. See table 1 for definitions of the abbreviations. B) Endothelial cell biology PCR array analysis of HPMVECs co-cultured with PAFLCs, MFLCs or MFLCs +Rapamycin. There were decreases in 15 and increases of 3 genes in ECs co-cultured with MFLCs in comparison to the expression in ECs co-cultured with PAFLCs (P < 0.05; n = 3). This result is related to 3 different patients out of six of co-culture or conditioned medium. See table 2 and 3 for the definitions. C) SMAD reporter signal in HPMVECs co-cultured with MFLCs. There was no statistical difference in the expression of SMAD reporter signal in ECs co- cultured with MFLCs in comparison to the expression in those co- cultured with PAFLCs treated with or without rapamycin. D) Accumulation of ROS in HPMVECs co-cultured with MFLCs. The decreased production of ROS has been detected in ECs co-cultured with MFLCs in comparison to the expression in those co-cultured with PAFLCs (P < 0.05; n = 3). Although there was a tendency that rapamycin treatment of ECs co-cultured with MFLCs reversed the decreased production of ROS, there was no statistical difference between them. Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 9 of 16 related genes (Figure 5A) (Table 1) and prevented the changes in expression in 11 of the 15 decreased and all three of the increased genes related to endothelial cell biology (Figure 5B) (Table 2, 3). There was no statistical diff erence in the expression of SMAD reporter signal in ECs co-cultured with MFL Cs with rapamycin (Figure 5C). Although rapamycin treatment of ECs co-cultured with MFLCs seemed to reverse the decreased produc- tion of ROS (Figure 5D), there was no statistical differ- ence between them. Confocal microscopy showed that the ECs co-cultured with MFLCs that were treated with rapamycin expressed LC3. Although the formation of autophagosomes (green punctate structures) was not detected in ECs co- cultured with MFLCs (Figure 6B), it was detected in these cells when they were treated with rapamycin (Fig- ure 6C). In the ECs co-cultured with MFLCs, the co- localization of Mitotracker red and SOD-2 was lost, indicating that the mitochondrial reticulum is disru pted (Figure 6E, 2M). However, the mitochondria in the ECs co-cultured with MFLCs that were treated with rapamy- cin form an intricate, filamentous network, in which SOD-2 and Mitotracker red are tightly co-localized (Fig- ure 6F, I). Discussion EnMT is a term which has been used to describe the process through which ECs lose their endothelial Table 1 Autophagy PCR array Biological process description Gene name Gene symbol Public ID P-value Autophagy Machinary Components: Genes Involved in Autophagic Vacuole Formation Autophagy/beclin-1 regulator 1 AMBRA1 NM_017749 0.00308 Autophagy Machinary Components: Genes Involved in Autophagic Vacuole Formation Genes Responsible for Protein Targeting to Membrane/ Vacuole Genes Responsible for Protein Transport Genes with Protease Activity ATG4 autophagy related 4 homolog D (S. cerevisiae) ATG4D NM_032885 NM_017749 0.01167 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis B-cell CLL/lymphoma 2 BCL2 NM_000633 0.000727 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis BH3 interacting domain death agonist BID NM_001196 0.047933 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4) CDKN2A NM_000077 0.044888 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Cathepsin B CTSB NM_001908 0.010802 Regulation of Autophagy: Chaperone-Mediated Autophagy Heat shock protein 90 kDa alpha (cytosolic), class A member 1 HSP90AA1 NM_001017963 0.037151 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Huntingtin HTT NM_002111 0.033212 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Co-Regulators of Autophagy and the Cell Cycle Interferon, gamma IFNG NM_000619 0.017749 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Insulin-like growth factor 1 (somatomedin C) IGF1 NM_000618 0.017282 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Insulin INS NM_000207 0.045037 Autophagy Machinary Components: Genes Involved in Autophagic Vacuole Formation Microtubule-associated protein 1 light chain 3 beta MAP1LC3B NM_022818 0.011251 Regulation of Autophagy: Co-Regulators of Autophagy and Apoptosis Autophagy in Response to Other Intracellular Signals Protein kinase, AMP-activated, alpha 1 catalytic subunit PRKAA1 NM_006251 0.005633 Autophagy Machinary Components: Genes Involved in Autophagic Vacuole Formation Regulator of G-protein signaling 19 RGS19 NM_005873 0.021592 Regulation of Autophagy: Autophagy in Response to Other Intracellular Signals Ribosomal protein S6 kinase, 70 kDa, polypeptide 1 RPS6KB1 NM_003161 0.024072 Regulation of Autophagy: Autophagy in Response to Other Intracellular Signals Transmembrane protein 77 TMEM77 NM_178454 0.019285 Regulation of Autophagy: Autophagy in Response to Other Intracellular Signals UV radiation resistance associated gene UVRAG NM_003369 0.016479 Functional classification of low expressed genes in co-cultured HPMVECs with MFLCs in comparison to PAFLCs Sakao et al. Respiratory Research 2011, 12:109 http://respiratory-research.com/content/12/1/109 Page 10 of 16 [...]... Lin GY, Yuan JX: Multipotent mesenchymal progenitor cells are present in endarterectomized tissues from patients with chronic thromboembolic pulmonary hypertension Am J Physiol Cell Physiol 2010, 298:C1217-C1225 Maruoka M, Sakao S, Kantake M, Tanabe N, Kasahara Y, Kurosu K, Takiguchi Y, Masuda M, Yoshino I, Voelkel NF, Tatsumi K: Characterization of myofibroblasts in chronic thromboembolic pulmonary. .. Rapamycin inhibits hypoxia-induced activation of S6 kinase in pulmonary arterial adventitial fibroblasts [36], suggesting the possibility that there may be a therapeutic benefit in PAH Moreover, rapamycin has an anti-proliferative effect on pulmonary arterial SMCs derived from endarterectomized tissues of CTEPH patients [37] In this study, we demonstrated that rapamycin reversed the decrease in autophagy... Zurita-Martinez SA, Cardenas ME: Tor and cyclic AMP-protein kinase A: two parallel pathways regulating expression of genes required for cell growth Eukaryot Cell 2005, 4:63-71 doi:10.1186/1465-9921-12-109 Cite this article as: Sakao et al.: Endothelial-like cells in chronic thromboembolic pulmonary hypertension: crosstalk with myofibroblastlike cells Respiratory Research 2011 12:109 Submit your next... dysfunction in ECs in patients with idiopathic PAH, similar to the SMCs in PAH [33] The existence of mitochondrial disorder/dysfunction in commercially available pulmonary microvascular ECs co-cultured with MFLCs in CTEPH and ECs in PAH, may support the similarities in the microvascular remodeling in the two disease Although several protein kinases regulate autophagy, the mammalian target of rapamycin... Group in Europe (VITAE) Venous thromboembolism (VTE) in Europe The number of VTE events and associated morbidity and mortality Thromb Haemost 2007, 98:756-764 Hoeper MM, Mayer E, Simonneau G, Rubin L: Chronic thromboembolic pulmonary hypertension Circulation 2006, 113:2011-2020 Moser KM, Bloor CM: Pulmonary vascular lesions occurring in patients with chronic major vessel thromboembolic pulmonary hypertension... autophagy was found in both ELCs (Figure 2K) and human pulmonary microvascular ECs co-cultured with MFLCs (Figure 6B) compared to the expression in human pulmonary microvascular ECs cocultured with pulmonary arterial fibroblast-like cells (Figure 6A), thus suggesting that in these cells, protein synthesis predominates over protein degradation Moreover, the decreased expression of cell death-related genes indicated... difference in the expression of SMAD reporter signal in ECs co-cultured with MFLCs in comparison to the expression in those co-cultured with pulmonary arterial fibroblast-like cells (Figure 5C) A recent study provides evidence that Ras/ MAPK, via TGF-b1 signaling, mediates completion of EnMT in a bleomycin model of pulmonary fibrosis [28] However, an endothelial cell biology PCR array in this study demonstrated... cell biology (Figure 5B) (Table 2, 3), thus suggesting that rapamycin (as an anti-proliferative agent) has beneficial therapeutic effects, not only on pulmonary arterial SMCs, but also on pulmonary arterial ECs which exist in the close contact with MFLCs, in the patients with CTEPH However, because rapamycin may act on the proliferation rate of MFLCs more than pulmonary arterial fibroblast-like cells [37],... rapamycin (mTOR), which negatively regulates the pathway in organisms from yeast to man, is the best characterized [15] Rapamycin is an inhibitor of mTOR and an anti-proliferative immunosuppressor that arrests cells in the G1 phase of the cell cycle [34] Rapamycin is used clinically in cardiovascular medicine as an anti-proliferative agent applied to coronary stents to reduce local restenosis [35] Rapamycin... may be stimulated (Figure 5A) This inactivation could benefit cancer cells Recently several genetic links between autophagy defects and cancers have been shown, providing increasing support for the concept that autophagy is a genuine tumor suppressor pathway [29] Signaling pathways that regulate autophagy overlaps with those that regulate tumorigenesis [16] This study has shown that human pulmonary . Open Access Endothelial-like cells in chronic thromboembolic pulmonary hypertension: crosstalk with myofibroblast-like cells Seiichiro Sakao 1* , Hiroyuki Hao 2 , Nobuhiro Tanabe 1 , Yasunori Kasahara 1 ,. MM, Lin GY, Yuan JX: Multipotent mesenchymal progenitor cells are present in endarterectomized tissues from patients with chronic thromboembolic pulmonary hypertension. Am J Physiol Cell Physiol. Rubin L: Chronic thromboembolic pulmonary hypertension. Circulation 2006, 113:2011-2020. 6. Moser KM, Bloor CM: Pulmonary vascular lesions occurring in patients with chronic major vessel thromboembolic

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Mục lục

  • Abstract

    • Background

    • Methods

    • Results

    • Conclusions

  • Background

  • Methods

    • Cell lines and reagents

    • Immunofluorescence staining

    • Double immunohistochemical staining

    • ELISA (Enzyme-Linked ImmunoSorbent Assay)

    • Human pulmonary microvascular ECs in the conditioned medium

    • Co-culture of human pulmonary microvascular ECs and MFLCs

    • Magnetic cell sorting (MACS)

    • Total RNA isolation and Quantitative measurement

    • PCR array analysis

    • SMAD reporter assay

      • Reactive oxygen species (ROS) assay

    • Statistical analysis

  • Results

    • The cellular composition of endarterectomized tissue from CTEPH patients

    • Phenotypic alteration of ELCs

    • Transitional cells in endarterectomized CTEPH tissue

    • Decreased expression of Autophagic marker LC3 (microtubule-associated protein1 light chain 3; MAP1LC3), abnormal mitochondria, and decreased expression of superoxide dismutase (SOD)-2 in ELCs

    • Phenotypic alteration of human pulmonary microvascular ECs is induced by MFLCs-conditioned medium

    • Expression of TGF-β1 protein in the conditioned medium

    • Phenotypic alteration of human pulmonary microvascular ECs co-cultured with MFLCs

    • Autophagy PCR array analysis of human pulmonary microvascular ECs co-cultured with MFLCs

    • Autophagic marker LC3 expression in human pulmonary microvascular ECs co-cultured with MFLCs

    • Abnormal mitochondria and decreased expression of superoxide dismutase (SOD)-2 in human pulmonary microvascular ECs co-cultured with MFLCs

    • Endothelial cell biology PCR array of human pulmonary microvascular ECs co-cultured with MFLCs

    • SMAD reporter signal in human pulmonary microvascular ECs co-cultured with MFLCs

    • Accumulation of ROS in human pulmonary microvascular ECs co-cultured with MFLCs

    • Rapamycin treatment

  • Discussion

  • Conclusions

  • Acknowledgements

  • Author details

  • Authors' contributions

  • Competing interests

  • References

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